Method for preventing postsplenectomic syndrome in experiment

FIELD: medicine, surgery, transplantology.

SUBSTANCE: embryonic spleen should be sampled, washed in nutritive medium № 199 to be placed into fresh medium № 199 to obtain homogenate in teflon homogenizer followed by centrifuging; then one should isolate the upper, medium and inferior layers, suck off medium layer and the upper part of inferior layer; the cell mixture obtained should be diluted in nutritive medium № 199 to be then introduced by injections into mesentery of small intestine or rectus muscle of abdomen. The present innovation favors the activation of immune system in patients undergone splenectomic operation and in those in case of surgical immunodefficient state due to high functional and regenerating activity of transferred embryonic splenic cells.

EFFECT: higher efficiency of prophylaxis.

6 dwg, 2 tbl

 

The present invention relates to medicine, in particular to surgery and transplantation, and can be used in surgical treatment of diseases and injuries of the spleen to prevent postsplenectomy syndrome and in the remote period after splenectomy.

The prototype of the present invention is a method autotransplantation of splenic tissue (Mironenko O. with co. Morphological justification for autotransplantation of spleen tissue //Medical business. - 1985. No. 11. - P.71-74). This method is autopijaca transplantation of splenic tissue in the mesentery of the small intestine or large pocket seal in the form of pieces with sizes up to 5 mm or minced. The disadvantages of this method are low functional activity of the transplanted tissue, the possibility of purulent-inflammatory complications, some contraindications to the use of this technique (diffuse peritonitis, massive bleeding). In addition, this method is applicable only intraoperative, when forced splenectomy.

The technical result is achieved by the use of the invention is the improvement of the functional state of the immune system in splenectomised patients, and in patients with surgical immunodeficiency States (sepsis).

This technical result of the temperature is raised to those what does the fence spleen fetal rat line “Wistar”, which is washed in a nutrient medium No. 199, then placed in fresh medium No. 199, get the homogenate in a Teflon homogenizer, and subjected to his centrifugation, there are the upper, middle and lower layers, sucked off the middle layer and the upper part of the lower layer is dissolved in a nutrient medium No. 199, then the resulting homogenate was injected injected in the mesentery of the small intestine or in the straight muscle of the abdomen.

The recovery method of the splenic tissue is carried out as follows. By laparotomy taking germ, remove the spleen of fetus, washed nutrient medium No. 199, then placed in fresh medium No. 199, in order to obtain a homogeneous mass by mechanical mastication in the homogenizer get the homogenate, which is poured into two identical tubes with an equal amount of content. Then these tubes centrifuged. Then the contents of the tubes is divided into three layers: top, middle, bottom. Each layer is subjected to a thorough morphological study on the qualitative study, quantitative composition of the layers, the viability and functional activity of cellular structures in them. Sucked off the middle layer and the upper part of the lower layer is dissolved in a nutrient medium No. 199, the lead injection in the mesentery of the small intestine or in the straight muscle of the abdomen of the adult rat.

Example. Was allocated 5 spleens embryos from a single female. After mechanical cleaning of the splenic tissue was washed in culture medium No. 199 and placed in fresh medium No. 199 (5 ml). The homogenate was obtained in a Teflon homogenizer. The obtained substrate was centrifuged in 2 beakers 2.5 ml for 30 minutes at a speed of 1500 Rev/min After which homogenized divided into 3 layers. Each layer in a separate flask and painted 1) Romanovsky-Giemsa, 2) Trifanova blue (dye, detecting dead cells) at a concentration of 0.4% solution. Morphological study showed that in the upper (2.5 ml) layer, almost no cells or cellular structures, on average (1 ml) is the main number of live splenocytes (90-95%) and a small number (5-10%) of the cell structure, lymphocytes, macrophages and other at the bottom (0.5 ml) lymphocytes, macrophages, cellular structure and in a small number of splenocytes.

The experiment was conducted on 35 adult white rats (m=250-320 g). Under ether anesthesia was performed upper median laparotomy. In the mesentery of the small intestine (slowly, insulin syringe) was administered 0.5 to 0.8 ml of a solution of the obtained homogenate. The injection site was marked nodal ligature. After manipulation produced layer-by-layer suturing.

With the introduction of homogenate in a direct muscle of a stomach to the anterior abdominal wall shots the Gali area on paramedicine line in the middle part of the abdomen. Then in the prepared area produced injection up to 1 ml in a straight muscle of the abdomen.

Postoperative mortality is not observed. The animals began to eat from the first day after surgery, were active, there were no fatal outcomes.

The operated animals were divided into 3 groups: the first group was scored after 10, second 20, third 30 days after transplantation. To assess the functional state of the transplanted tissue performed histological and histochemical studies. From the transplant was removed pieces of tissue sizes 1×1 see the recovered fragments were fixed in 10% formalin solution and after the corresponding histological preparation and cooking slice thickness of 7 μm were stained with hematoxylin-eosin. Just cooked 350 specimens. The filter function of the transplanted splenic tissue was evaluated by Perls reaction that detects the presence of hemosiderin, which is the intermediate product of elimination of erythrocytes. Pieces of the graft in the cryostat at -20°cooked 60 drugs thickness of 7 μm.

Morphological study of drugs is illustrated by the following figures:

Figure 1. Fragment of splenic tissue after 10 days after transplantation in the mesentery of the small intestine. The color of gematik the Yichang-eosin. Microphoto. OK. 10,Ob.

Figure 2. The white pulp of the graft spleen through 20 days after transplantation of embryonic tissue. Paint hematoxylin-eosin. Microphoto. OK about. 20.

Figure 3. The increase in the number of lymphoid cells in the red pulp through 20 days after transplantation of embryonic tissue. Paint hematoxylin-eosin. Microphoto. OK. 10, vol. 40.

Figure 4. The graft spleen through 30 days after the experiment. Paint hematoxylin-eosin. Microphoto. OK. 7, vol. 20.

Figure 5. Sideroblastic transplant within 30 days after the experiment. The Reaction Of Perls. Microphoto. OK. 10, vol. 40.

6. Lymphoid follicles (white pulp of the spleen) in striated muscle tissue within 10 days after transplantation. Paint hematoxylin-eosin. Microphoto. OK. 10, vol. 20.

Within 10 days after transplantation of splenic tissue in the mesentery of the small intestine texture was determined by a dense formation of a dark red color, visual signs of inflammation are not checked.

In loose connective tissue, primarily determined by large aggregations of lymphoid tissue corresponding to the white pulp of the spleen. It presents the lymphatic nodules, periarterial lymphatic sheaths, and the marginal zone. Lymph follicles are located on the periphery of the periarterial what about the lymph vagina. Periarterial lymphatic vagina surrounds the Central artery. The composition of this lymphoid tissue graft composed of lymphocytes, macrophages and reticular cells (figure 1). The marginal zone is a narrow layer of lymphocytes around knots and periarterial lymphatic sheaths.

The red pulp presents venous vessels and splenic bands, where small amounts are found erythrocytes. Lymphoid tissue located in the loops reticular tissue, occupies a much larger space. Around the red and white pulp of the spleen in the surrounding loose connective tissue scattered in small size aggregations of lymphoid tissue, all of them are round or oval. Some accumulations of lymphoid cells may be located in close proximity to blood vessels between the lipocytes and large lymphoid formations. Around small clusters of lymphoid cells include fibroblasts and lipocytes are single spaced lymphocytes and macrophages.

Within 20 days after transplantation of splenic tissue also define all the structures of the spleen. The white pulp consists of lymphatic nodules, periarterial lymphatic sheaths, and marginal zone. Is determined by the eccentrically located Central artery. Cells and lymphoid tissue of the white pulp of the spleen is distributed evenly, however, in the lymph nodules centers reproduction cannot be detected. The red pulp is characterized by an increase in the number of lymphoid cells located between reticular cells, however, reticular cells have spread view (figure 2). In the surrounding connective tissue around the white and red pulp scattered lymphoid aggregations of a variety of shapes and sizes. There are clusters of large size and many single located lymphoid cells. Around the graft is well developed network of blood vessels like capillaries and larger blood vessels of moderate blood (figure 3).

Within 30 days after the experiment in the transplant also determined by the presence of white and red pulp. The white pulp consists of lymph follicles, and in the red pulp of many lymphocytes and macrophages, located among the “spread” reticular cells. Around the white and red pulp of the graft spleen, in the surrounding loose connective tissue many different size of lymph follicles. Some of them are located in the immediate vicinity of large blood vessels. At the same time is determined by a diffuse proliferation of lymphoid cells from both white and red pulp, and from emerging foci of lymphoid tissue located in the loose match the nutrient tissue (figure 4). This is a single located lymphoid cells include fibroblasts and lipocytes. The dynamics and nature of the appearance of foci of lymphoid tissue around the graft are presented in table 1.

At various times after transplantation of embryonic splenic tissue in the mesentery of the small intestine is determined by the varying intensity of the response to iron in the macrophages of the red pulp. Macrophages were placed in small groups or diffuse. The white pulp of the graft remained intact to the iron. Hemosiderin was determined in the form of granules of different size (figure 5). In most cases, the granules of hemosiderin in the cytoplasm of sideroblasts evenly distributed, however, met macrophages with large granules.

Within 10 days after the introduction of splenic tissue in a straight muscle of the abdomen among the fibers of the striated muscle tissue of the anterior abdominal wall is a round shaped accumulation of lymphoid tissue, consisting of lymphocytes and macrophages located among reticular cells (6). Lymphoid cells have dense location, determined by the presence of the Central artery. Around a large accumulations of lymphoid cells are freely located lymphoid cells. They extend along the muscle fibers in permisi front abdominal muscles. Meet slightly the size of the accumulations of lymphoid cells. All of the above patterns do not have signs of inflammation.

After 20 and 30 days is determined by the presence of large lymphoid nodules, consisting of lymphocytes and macrophages. Around lymphoid nodules in the loose connective tissue between muscle fibers are quite often different sizes lymphoid aggregations. While lymphocytes and macrophages are tight. All cells have a clear outline of the core and shell. The chromatin of the cells is very dense, the cytoplasm of cells basophilia. Free arranged lymphoid cells penetrate far enough away from the main focus of lymphoid tissue, cells penetrate between muscle fibers in permisi.

It is conceivable that the formation of new foci of blood around the graft and active functioning of the cells of the graft occurs under the influence of stimulants lymphoid nodules graft spleen.

In the transplanted splenic tissue is determined by the functional activity of macrophages of the red pulp, perform the filtration function, absorbing damaged erythrocytes. This fact may serve to confirm the possibility of performing the transplanted splenic tissue neutralizing function. However, we cannot exclude the possibility of migration of macrophages from adjacent tissues of the donor and p is the administrative degradation products of hemoglobin from the network vessels located around the graft.

To determine the functional activity of the transplanted splenic tissue we performed immunological research. Research ability to synthesis hemagglutinins antibodies, reflecting the protective function of the body, carried out within 2-3 months after surgery, according to the literary sources of this period the activity of an adequate immune reactions can be completely recovered. For reliability of the study the rats that were transplanted splenic tissue, operations simultaneously produced splenectomy.

After the 3-fold immunization operated rats was determined indicators of immune response by titration of hemaglutinin xenogeneic erythrocytes. This was determined by the ability of immune cells to the synthesis hemagglutinins antibodies. Immunization was performed according to the scheme; 1st immunization 2 weeks after surgery; 2nd immunization after 1 week after the 1st immunization, 3rd immunization 3 weeks after the 2nd immunization. Survey data are given in table. 2. For comparison we have selected a group of animals after laparotomy and splenectomy.

As can be seen from the obtained results, after the first immunization was observed significant differences (p<0.05) in the immune response to centuries is Denia antigen in all groups of animals. After the 2nd, especially after the 3rd, immunization of animals undergoing laparotomy, the titer hemaglutinin significantly increases, reaching a maximum value by the end of the 4th week. The titer hemaglutinin in animals after transplantation of splenic tissue and splenectomy was relatively lower than in animals after laparotomy throughout the study period. However, it should be noted reliable difference in the dynamics of the rise in the titer of antibodies in rats with transplanted splenic tissue and fully remote spleen (p<0,05). Therefore, antibody productive function suffers less in rats after transplantation of splenic tissue compared with rats after splenectomy.

Experiments were carried out on 35 rats. Compared with the prototype in the present invention provides a high functional activity of the transplanted cells, their high regenerative ability, there are all the signs of the emergence of new foci of hematopoiesis. Furthermore, the reduced risk of immunocomplex due to incomplete immune differentiation of embryonic tissue. For the period of embryonic development have peak activation of the so-called growth factor, accelerating functional properties of splenocytes. Conducted immunological studies have demonstrated significant compensat the Yu immunological capabilities of animals with transplanted splenic tissue compared with splenectomised animals. Equipment operation is simple, requires no special tools, preliminary special training of medical personnel, the method can be used not only in specialized, but in General surgical wards.

Table 1.

Trends in the number and size of lymphoid tissue in the mesentery of the small intestine.
Survey duration (days)The size of the accumulations of lymphoid tissue around the graft
 SmallAverageLarge
10Rare clusters (1-2-3 focus)--
203-4 hearth3-4 hearth1-3 hearth
305-7 foci5-6 foci3-5 lesions
Tablica.

Antibody titer operated rats after operations (M+m) (titer before surgery 1:32+1:1280)
The follow-up periodComparable groups of animals
 laparotomy splenectomyafter transplantation
1 week after the 1st immunization1:220-1:16001:58-1:2681:120-1:110
1 week after the 2nd immunization1:1022-12001:240-1:18001:240-1:180
2nd week after the 2nd immunization1:1200-1:181101:560-1:1410011:830-1:215
Is the 3rd week after the 2nd immunization1:2350-1:181201:560-5-1:1233401:950-1:170
1 week after the 3rd immunization1:8200-1:246001:980-1:193001:1660-1:22

cont. table 2
2nd week after the 3rd immunization1:10220-1:24701:1034-1:222001:2380-1:20
Is the 3rd week after the 3rd immunization1:10220-1:32401:1840-1:181101:4092-1:22
The 4th week after the 3rd immunization1:16350-1:48201:4086-1:486001:4100-1:17
8-and the week after the 3rd immunization 1:8186-1:42201:2400-1:192201:2400-1:19

The way to prevent postsplenectomy syndrome by transplantation of splenic tissue in the mesentery of the small intestine, characterized in that the taking of the spleen in the embryo, which is washed in a nutrient medium No. 199, then placed in fresh medium No. 199, get the homogenate in a Teflon homogenizer, and subjected to his centrifugation, there are the upper, middle and lower layers, sucked off the middle layer and the upper part of the lower layer is dissolved in a nutrient medium No. 199, injected injected in the mesentery of the small intestine or the straight muscle of the abdomen.



 

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